HPTLC

For the analysis of botanical materials and

medicinal plants
Eike Reich

CAMAG Laboratory
Sonnenmattstrasse 11
4132 Muttenz / Switzerland
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What will be discussed?

1. What exactly is HPTLC?

2. The elements of a standardized HPTLC methodology

3. Changing TLC methods into HPTLC methods

4. Development and validation of ID Methods

5. How new developments in HPTLC may affect the analysis of plants

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Introduction
 Thin-layer chromatography (TLC) always was and still remains an
important tool for the analysis of plants.
 Today there are two principal applications in this context: research and
quality control. Both benefit from the advantages of the planar off-line
principle and also in particular from low cost, simplicity, and flexibility.
 For decades TLC is integral part of monographs for medicinal plants in
all pharmacopoeias and the primary method of identification.
 Growing expectation regarding performance charateristics have brought
TLC methods to the limits.
 Since the turn of the century pharmacopoeias recognize the technical
progress in instrumentation and improvements offered by high
performance plates.
 Most recently HPTLC is being discussed as alternative to classical TLC.
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The planar off-line principle

the plate
application

development

detection and quantitative evaluation

documentation FLEXIBILITY
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But what about reproducibility?

The central problem:

 When two (or more) labs do the „same“ or think that they are doing the
same, the results are not necessarily equal.
 Reason is the general method description in the Pharmacopoeias (EP
2.2.27, USP <201>, <621>, PhPRC ap. VI) which define „suitable”
equipment and give ranges instead of values.
 JPXV 2.03 is still centered around self made 20x20 TLC plates !
 A table (EP) or a result description (USP, JP) can only define the most
Most text books are even worse …
important aspects of a TLC chromatogram. That leaves room for
interpretation. An atlas (PhPRC) provides clear guidance.
 Example: how can a color be described correctly?
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What is blue?

And how about the

color blind...
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PhEur: Possible choices in methodology

 TLC layer  HPTLC layer

 Manual application  Automatic application

 Transparent container  Automatic Developing

(Pickle jar?) Chamber

 UV-Lampe (?)  Scanner

 Manual spraying /  Automatic immersion /

immersion spraying
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Identification of Acanthopanax
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Identification of Peonies
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Identification of Fleece flower

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Goal:
International standardization of HPTLC
 What
 HPTLC definition
 Methodology
 Equipment
 Why
 Reproducibility of results
 Validity of official methods
 Quality assurance in a globalized world
 Quality of published research
 How
 International collaboration
 Top down
 Publication
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TLC or HPTLC
 Pharmacopoeias see difference primarily in the plate
yet assume similar results

HPTLC plate

Average plate height [μm]

e
at
pl
C
TL
HP
TLC plate

0 10 20 m
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Comparison TLC-HPTLC of flavonoids

TLC plate 20 x 20 cm
(135 mm)
HPTLC plate 20 x 10 cm
(60 mm)
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TLC or HPTLC?
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What is TLC?
 Chromatography for the poor (cheap)
 Simple manual chromatography for everyone
(students?)
 Rapid
 Flexible
 Reference and test solution side by side
 “Just” qualitative, preliminary estimation at best

 Unpredictable
 Unreliable

 Manual technique, simple instruments, TLC plates

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What is HPTLC?

High Performance Thin-Layer Chromatography

TLC for the 21st century A new concept
 Instrumental TLC
 Suitable instruments
 Application
 Development
 Scientific basis
 Documentation
 Densitometry  Standardized methodology
 Truly „plug and play“
 Fully cGMP compliant  Validated methods
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“HPTLC” on TLC plates?

 What is the point?
 Saturation 1h vs. 20 min
 Twice (10x) the solvent volume
 3x the developing time (15 vs. 6 cm)
 Same cost per plate (20x20 vs. 10x10 cm)
yet
 Less resolution
 No control of the development process
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Standardization of methodology
 Plate setup and handling
 Sample application (as band)
 Chamber geometry and saturation
 Humidity control
 Developing distance
SOP
 Derivatization procedure
 Documentation (electronic images)
 Evaluation
A Standardized Approach to Modern High-Performance Thin-Layer Chromatography
(HPTLC). Reich, E., Schibli, A., (2004) J. Planar Chromatogr. 17, 438-443
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SOP for HPTLC

 Should be the basis for all work (in participating labs)

 Applies to all methods

 All deviations need to be recorded

 Our SOP is in full compliance with PhEur, USP, ChP

Available at: www.camag-laboratory.com (homepage)

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The basic HPTLC setup

Application Development Derivatization Documentation

NOTE
: stand
a rdiza
tion d
Only oes n
agree ot req
m e nt uire s
abou pecif
t all p ic eq
aram uipm
eters ent !
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Standardized basic equipment

 Clear specification of HPTLC plates (type, format, manufacturer)

 Software control for “absolute” reproducibility of all parameters

 Independence from environmental factors (humidity !)

 Eliminating human factors

 Emulation by “manual” operation still possible to a certain degree

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Sources of methods
 European Pharmacopoeia (EP)

 New monographs feature TLC and HPTLC in parallel

 British, French, German, Swiss Pharmacopoeias

 Offer specific monographs not found in EP

 The USP Dietary Supplement Compendium

 TLC and state of the art HPTLC

 Chinese Pharmacopoeia

 HPTLC atlas as a supplement of 2005 ed.

 Japanese Pharmacopoeia

 Only simple TLC

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More sources of methods

 American Herbal Pharmacopoeia (19 monographs)

 Quality Standards of Indian Medicinal Plants

(8 volumes)

 Indian Herbal Pharmacopoeia (54 monographs)

 Quality Standards of Traditional Chinese Medicines

(Chinese only)

 Wagner, H. and Bladt, S. „Plant Drug Analysis“

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Converting existing TLC methods to HPTLC

Assumptions:
 Results on HPTLC and TLC plates are similar if
 No changes are made to chromatographic system
 Same equipment is used
 Original TLC method was optimized
 Due to the higher separation power HPTLC plates
 Usually give improved result
 Require shorter developing distance  less time
 HPTLC with no instruments looks bad, but so does TLC
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Converting existing TLC methods to HPTLC

Practical aspects (I)
 Do not change chromatographic system (chamber configuration, mobile
phase, stationary phase)
 Reduce application volume (generally) to 1/5 (typically 2 μL)
 Employ standardized methodology (based on SOP):
 Fixed (x, y) application positions, (e.g.) 8 mm bands
 Use 60 mm developing distance
 Fixed drying time and temperature
 Use dipping instead of spraying if possible
 Fixed waiting times between derivatization and evaluation
 Obtain multiple images (if possible) of plate (e.g. UV 254, UV 366
prior to derivatization, and white light, UV 366 after derivatization
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Converting existing TLC methods to HPTLC

Practical aspects (II)
 Use same samples on TLC and HPTLC
 Evaluate whether changes in the result can
 Fall under “additional weak zones may be seen”
 Are due to natural variability of plant
 Color description is always subjective: e.g.
blue, bluish
blue white
blue green, etc.
 Rf is predictable only for validated methods!
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Example: USP method for chamomile

TLC

HPTLC
application volume 2 μL
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Developing an ID method from scratch

 Review literature for related plants

 Obtain multiple samples form different accessions

 Obtain samples of related plants and known adulterants

 Optimize sample preparation and detection

 Avoid toxic solvents

 Start with silica gel, select mobile phase

Reich, E., Schibli, A.:HPTLC for the analysis of medicinal plants, chapter 5, Thieme 2007
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Validation of qualitative methods

Validation protocol

Validated method
Method selection

Optimization

Robustness
Specificity

Precision
Stability

Validation of high-performance thin-layer chromatographic methods for the identification of botanicals in a

cGMP environment. Reich, E., Schibli, A., DeBatt, A. (2008) J. AOAC Int. 91, 13-20.
Envisioning the Future...
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Methods for identification of plants

 SOP is the basis for an HPTLC method template (instruments)

 software template

 A (PhEur, USP, etc.) monograph or validated methods is basis for

HPTLC method document Method for identification of Thyme

 Result table/description is replaced by a reference image

 Corresponding instrument methods are derived from software template

 instrument method

 Results on plate are qualified by a System Suitability Test (SST)

 Comparison of results with reference images

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Thyme leaf plates 1 - 3

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Thyme leaf
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Goal:
The CAMAG HPTLC method collection

 Identification of 150 (+) plants from East and West

 Predictable results everywhere

 Convenient comparison against reference images

 Compatible with (current and future) harmonized

description of HPTLC

 Based on ATS4, ADC2, Visualizer and Software

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Goal:
CAMAG Laboratory Network

 Collaboration on development and validation of

HPTLC methods for identification of plants

 Setting global quality standards for HPTLC

 Providing training and research opportunities

 Offering analytical services for customers

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Who is part of the network?

 CAMAG Laboratory Muttenz
 CAMAG Scientific Inc. Wilmington (NC)
 Shanghai University of TCM (Prof. Wang)

 University of Applied Sciences Wädenswil (Prof. Meier)

 University of Regensburg (Prof. Heilmann)
 University of Barcelona (Prof. Canigueral)
 University Sapienza Rome (Prof. Nicoletti)
 University of Graz (Prof. Bauer)
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Examples from other labs (HS Wädenswil)

Oswego tea
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Examples from other labs (Uni Regensburg)

Coptis rhiz.
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A new software concept !

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Three channels per track

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PCA (Master thesis R. Ambühl, Uni Basel)

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HPTLC as research tool

 Evaluation of column fractions

 Identification by MS

 Screening for bio-activity

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Diploma thesis M. Meier Uni Graz

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Diploma Thesis R. Vizzini (CAMAG)
Identification of alkaloids in Sophora flavescens
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Identification of alkaloids in Sophora flavescens

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BioLumineX - BioLuminizer
detection: bioluminescence Vibrio fischeri 06699

A B C D E F G H I np np np
detection: derivat. anisaldehyde / 366 nm

A - I : chamomile oil np : 4-nitrophenol

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Bio-assays: DPPH
Anti-oxidative properties of esential oils

Silica gel 60 F 254

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Toluene : ethyl acetate
95:5

1: carvacrol methylester; 2: thymol; 3: carvacrol; 4: thyme oil; 5:sage oil; 6:neroli oil; 7: lemon, oil; 8: peppermint oil;
9: rosemary oil; 10: chamomile oil; 11:sweet orange oil; 12: manuka oil; 13: tea tree oil; 14: pine oil; 15: niaouli oil;
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Bio-assays: DPPH
Anti-oxidative properties of flavonoids

Silica gel 60 F 254

Ethyl acetate, acetic
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 acid, formic acid, water
100:11:11: 27

1: chlorogenic acid; 2: quercitrin; 3: rutin; 4: ginkgo leaf extract; 5: St. John’s wort; 6: great mullein; 7: ribwort plantain; 8:
rosemary; 9: majoram; 10: basil; 11: thyme, 12: chamomile; 13; peppermint; 14: arnica; 15: birch; 16: hibiscus
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Thank you!

eike.reich@camag.com

www.camag-laboratory.com
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